Identification and Validation of RNA Silencing Associated Genes in Citrus sinensis

RNA silencing has been demonstrated to be involved in gene regulation through production of miRNAs and siRNAs that can be induced by biotic or abiotic stresses.   Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), and Argonaute (AGO) proteins are considered to be core components of the RNA silencing machinery.  These genes have been extensively studied in Arabidopsis thaliana, however, they have not been identified or validated in the agriculturally significant crop Citrus sinensis (Sweet orange).  In the present study, DCLs, RDRs, AGOs, and other silencing associated genes were identified through protein homology with Arabidopsis using bioinformatics tools made available since the recent release of the C. sinensis draft genome (JGI v.1.0).  Arabidopsis possesses 4 DCL, 6 RDR, and 10 AGO proteins and we identified 5 DCLs, at least 5 RDRs, and 10 AGOs in C. sinensis.  Phylogenetic comparisons between C. sinensis and Arabidopsis genes uncovered multiple paralogs in C. sinensis related to RDR1, RDR6, DCL2, AGO5 and AGO9, while lacking definitive AGO2 and AGO3 proteins. Gene expression was validated using RT-PCR from cDNA of C. sinensis (var. Hamlin) seedlings; select RT-PCR products were cloned and sequenced to determine amplicon specificity and authentication of the silencing gene sequence in the draft genome.  Computational analysis of publicly available high throughput RNA sequencing (RNA seq) of healthy and Huanglongbing diseased C. sinensis independently validated the expression of the silencing genes.  Furthermore, this RNA seq analyses reveals differential and distinct expression patterns among the twenty silencing associated genes.